CSB346 Assignment 2 Notes

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University of Toronto St. George
Cell and Systems Biology
John Peever

CSB346 Tutorial Assignment 2 Notes (March 14, 2013) Objective - They injected a virus so they could transduce ChR2. - They used a virus (AAV2-DIO). The virus delivered ChR2. Viral constructs - If the control existed, it would turn red, but they are not going to fire when you shine them with light. The viral construct transduce ChR2 in the neurons so they can be activated by light. o This is not the control. ePet-cre - They expressed the viral vector in ePet-cre mice. It is a specific transgenic mouse line where it has an ePet-cre promoter region. This means that ePet-cre (Pet-1) is a transcription factor that allows specific targeting because this particular transcription factor is involved with the development of serotonergic neurons. Only serotonergic neurons have the ePet-cre transcription factor. This is a transgenic mouse line that is expressing ePet-cre. They selectively targeted serotonergic neurons by using ePet-cre as the transgene because ePet-cre is specific to serotonergic neurons. - Cre-recombinase is the transgene that is only expressed in serotonergic neurons. Caudal raphe - The caudal raphe, which is not connected to the dorsal raphe, is composed of three parts (RM, RP, RO). They are all serotonergic. They are all close to each other, but they are all defined enough that they have different properties and they do different things. In this paper, they specifically target the RO because they want to know what the RO does. Figure 1 - This figure tells you where the RO serotonergic neurons project to. Over time, the virus will express ChR2 over the neuron. The longer you let that happen, the ChR2 will get expressed all the way down to the axons. The virus also expresses mCherry, which glows red, so you can look at where it shows red axons. You can show where those projections are coming from. - By letting the virus transduce long enough, all the axons will express ChR2 and the mCherry reporter gene. You can see that there are axons that project to numerous areas by looking at where mCherry is being expressed. - Figure 1 lists some of the areas that serotonergic RO neurons projected to. It doesn’t show you all of them. It tells you where all the projections went to in the actual text. Know where the axons project to in the text, not just relying on Figure 1. Figure 2 - They wanted to inject a virus and express it into just serotonergic neurons. - This figure tells you that when they try to transduce/inject ChR2 protein, they want to make sure that it was in the RO, and limited expression in the other two. It is limited to serotonergic neurons, but RP and RM are both serotonergic. They want to make sure that they are trying to prevent the spread going further than it needs to. - Most of the neurons that are expressing ChR2 are in the RO. You get a little bit in the other two, but the majority of them are in the RO. They can say with confidence that if they are going to shine light on them, then it is probably the RO neurons that is doing the majority of the effect. Figure 3 - This tells you how they optimized the signal on what kind of light they used. - There are different light sensitive proteins in the brain. You need to know how much light to give it. If you give too much light, you can cause heat damage. If you shine too much light, then is it caused by the light or heat? They optimized the signal (frequency and duration). - A o They fixed the frequency at 20 Hz. Then they tried it at various times. At 10 ms, you get an increase in diaphragm activity and an increase in breath frequency. When you shine it for 20 ms, you also get an increase, which is more or less the same as 10 ms. So shining at 10 ms is giving you optimal effects without going all the way to 20 ms. - B o This is the graph form of A. - C o They fixed the time so that they are firing at 20 ms. They changed the frequency. At 10 Hz and 20 Hz, unlike the 10 ms and 20 ms, there is a difference between the two. Maybe 20 Hz is better if you want to have the maximum effect for the light being used. - D o This is the graph form of C. - E o If you use the maximum (20 Hz, 20 ms) from A and C, it shows maximum effect. If you use these parameters, you find that it increases diaphragm amplitude (increases the depth of breathing), increases the frequency of breathing, and increases ventilation (amplitude*fR). - F-H o You do not need to know. When you turn on the light, frequency goes up. When you turn off the light, frequency still continues to go up before it starts going down. - Know what they optimized and chose to use. Figure 4 - This is an example of the authors being thorough. They used transgenic mouse lines that are specific to serotonergic neurons, so they know the virus can only be expressed in serotonergic neurons. - They wanted to prove that they are serotonergic neurons. He proved this by using a serotonergic antagonist, so that even if you were to excite serotonergic neurons, you wouldn’t see the effects. - A
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